1. Field of the Invention
The present invention relates to a method for determining total homocysteine.
2. Description of the Prior Art
Homocysteine, which is one of the metabolic intermediates in methionine metabolism, is reported to have vascular endothelial cytotoxicity and to be one of the risk factors for arteriosclerotic diseases independent from the other risk factors. It also has been evident that in addition to serious hyperhomocysteinemia (homocystinuria) caused by deficiency of homocysteine metabolic enzymes, moderate hyperhomocysteinemia is caused by a decrease in the metabolic enzyme activity due to abnormality of genes, renal insufficiency, aging, smoking, lack of exercise or the like (Jacobsen, Clin. Chem. 44:8(B), 1833-1843, 1998). Furthermore, hyperhomocysteinemia is also reported to be improved by taking vitamin B6, folic acid or the like (JAMA 270: 2693-2698, 1993). Therefore, not only for neonatal mass screening, but also for prevention of adult arteriosclerotic diseases or detection of vitamin deficiency diseases, a simple method for treating a large number of specimens is in demand.
Most of homocysteine in blood (99%) is present in the form of oxidized disulfide compounds (such as complex with protein, homocystine, cysteine-homocysteine) (Jacobsen, Clin. Chem. 44:8(B), 1833-1843, 1998). xe2x80x9cTotal homocysteinexe2x80x9d refers to the total amount of oxidized and reduced homocysteines, and in general, it is necessary to convert homocysteine in a sample to reduced homocysteine by a reducing agent in order to determine the total homocysteine.
High-performance liquid chromatography (HPLC) and immunoassay are usually employed to determine homocysteine. However, the HPLC apparatuses used in high-performance liquid chromatography are not commonly used in the clinical test, and it takes time, labor and cost to operate the apparatuses. In the immunoassay, although apparatuses are automated (Shipchandler, Clin. Chem., 41, 7, 991-994, 1995), determination is performed by combining a process for converting homocysteine to S-adenosyl-L-homocysteine by an enzyme reaction and a process for detecting it by an immunoassay, so that an apparatus used exclusively for this purpose is required.
Determination methods of homocysteine based on an immunoassay are proposed in Japanese Laid-Open Patent Publication (Tokuhyo) No. 9-512634 and (Tokkai) No. 10-114797. In the method disclosed in Japanese Laid-Open Patent Publication No. 9-512634, homocysteine is determined immunologically by chemically modifying the homocysteine to enhance the antigenicity, which requires a large number of processes and is complicated. Japanese Laid-Open Patent Publication No. 10-114797 discloses a method for determining homocysteine, but this method directly determines only the homocysteine bound to albumin, and does not determine the total amount of homocysteine. In this method, only about 70% of the entire homocysteine can be determined.
On the other hand, biochemical determination methods of homocysteine are disclosed in Japanese Patent No. 2870704, U.S. Pat. Nos. 5,998,191 and 5,885,767. The method disclosed in Japanese Patent No. 2870704 is characterized by allowing homocysteine in a sample that has been treated with a reducing agent to be in contact with adenosine and S-adenosyl-L-homocysteine hydrolase and evaluating the amount of adenosine in the residual mixture. However, in this method, an inhibitor of the S-adenosyl-L-homocysteine hydrolase is not used, and therefore it is necessary to perform determination in kinetic mode. Furthermore, this method has the problem that produced hydrogen peroxide cannot be led to a commonly used oxidative color-developing agent in the presence of a reducing agent that is used for a reduction process, which is an essential process for determining the total homocysteine. Therefore, an automatic analysis apparatus for general purposes cannot be used. However, these patent specifications fail to disclose any method to avoid these problems.
The methods disclosed in Japanese Laid-Open Patent Publication (Tokuhyo) No. 2000-502262, U.S. Pat. Nos. 5,998,191 and 5,885,767 are characterized by reacting homocysteine with homocysteine desulfurase, homocysteinase, or methionine-xcex3-lyase to detect the produced hydrogen sulfide, ammonia, or 2-oxobutyric acid. However, these methods have problems such as: requiring a large number of processes; employing a lead ion, which is a harmful heavy metal, for the detection of the hydrogen sulfide; and being affected by cysteine and methionine, which are structural analogs to homocysteine and contained in a biological sample in a larger amount than that of homocysteine.
Thus, the conventional methods of determining homocysteine have problems such as requiring a special apparatus and complicated operation and having insufficient sensitivity and specificity, so that a method for determining a trace concentration of homocysteine rapidly, simply and with high sensitivity has not been established yet.
The present invention provides a novel method for determining homocysteine contained in a biological sample or the like rapidly, simply and with high sensitivity, and a kit for use in this determination method.
The inventors of the present invention succeeded in detecting or determining homocysteine by (i) oxidizing the residual homocysteine cosubstrate, the produced homocysteine-converting enzyme product or an enzyme reaction product thereof in the presence of an SH reagent to produce hydrogen peroxide and detecting or determining the produced hydrogen peroxide by color development using an oxidative color-developing agent or (ii) reacting the residual homocysteine cosubstrate, the produced homocysteine-converting enzyme product or an enzyme reaction product thereof with a D-amino acid converting enzyme to produce an oxo acid and/or ammonia and detecting or determining the produced oxo acid and/or ammonia. With the method for determining homocysteine of the present invention, homocysteine in a biological sample, in particular in body fluids such as blood and urine can be detected and determined rapidly and simply.
The present invention is directed to a method for detecting or determining homocysteine in a sample including:
(a) reducing the homocysteine in the sample by a thiol compound,
(b) reacting the reduced homocysteine with a homocysteine-converting enzyme and a homocysteine cosubstrate, thereby producing a homocysteine-converting enzyme product, and
(c) detecting or determining the residual homocysteine cosubstrate, the produced homocysteine-converting enzyme product or an enzyme reaction product thereof by: (i) oxidizing the residual homocysteine cosubstrate, the produced homocysteine-converting enzyme product or an enzyme reaction product thereof in the presence of an SH reagent to produce hydrogen peroxide and detecting or determining the produced hydrogen peroxide by color development using an oxidative color-developing agent or (ii) reacting the residual homocysteine cosubstrate, the produced homocysteine-converting enzyme product or an enzyme reaction product thereof with a D-amino acid converting enzyme to produce an oxo acid and/or ammonia and detecting or determining the produced oxo acid and/or ammonia.
In one preferable embodiment, the homocysteine-converting enzyme in the step (b) is S-adenosyl-L-homocysteine hydrolase, and the homocysteine cosubstrate in the steps (b) and (c) is adenosine.
In one preferable embodiment, the step (c) of detecting or determining the adenosine is a step of detecting or determining the adenosine by reacting the adenosine with adenosine deaminase, phosphoric acid, purine nucleoside phosphorylase, and xanthine oxidase to produce hydrogen peroxide and detecting or determining the produced hydrogen peroxide by color development using peroxidase and an oxidative color-developing agent.
In a more preferable embodiment, the step (c) includes further reacting the adenosine with uricase.
In another more preferable embodiment, the homocysteine-converting enzyme in the step (b) is a methyltransferase using homocysteine as a methyl acceptor, and the homocysteine cosubstrate in the steps (b) and (c) is a methyl donor.
In one preferable embodiment, the methyltransferase is homocysteine methyltransferase, and the methyl donor is D-methionine methyl sulfonium.
In a more preferable embodiment, in the step (c), the homocysteine-converting enzyme product is D-methionine, and the D-methionine is determined by reacting it with a D-amino acid converting enzyme.
In a more preferable embodiment, the D-amino acid converting enzyme is D-amino acid oxidase.
In one preferable embodiment, in the step (c), the hydrogen peroxide produced by a reaction with the D-amino acid oxidase is detected or determined by color development using peroxidase and an oxidative color-developing agent.
In one preferable embodiment, the SH reagent is a maleimide derivative.
In another preferable embodiment, in the step (c), a decrease in NAD(P)H or an increase in NAD(P) is detected or determined by reacting the produced oxo acid and/or ammonia with dehydrogenase using NAD(P)H as a coenzyme.
In one preferable embodiment, in the step (c), the oxo acid and/or ammonia produced by a reaction with the D-amino acid oxidase is detected or determined.
In one preferable embodiment, in the step (c), a decrease in NAD(P)H or an increase in NAD(P) is detected or determined by reacting the produced oxo acid and/or ammonia produced by a reaction with the D-amino acid oxidase with dehydrogenase using NAD(P)H as a coenzyme.
In one preferable embodiment, the dehydrogenase is leucine dehydrogenase, and a decrease in NAD(P)H is detected or determined by reacting the produced oxo acid with the leucine dehydrogenase in the presence of ammonia and NAD(P)H.
In one preferable embodiment, the dehydrogenase is lactate dehydrogenase, and a decrease in NAD(P)H is detected or determined by reacting the produced oxo acid with the lactate dehydrogenase in the presence of NAD(P)H.
In one preferable embodiment, the dehydrogenase is glutamate dehydrogenase, and a decrease in NAD(P)H is detected or determined by reacting the produced ammonia with the glutamate dehydrogenase in the presence of 2-oxoglutaric acid and NAD(P)H.
In a more preferable embodiment, the dehydrogenase is lactate dehydrogenase and glutamate dehydrogenase, and a decrease in NAD(P)H is detected or determined by a reaction with the lactate dehydrogenase and the glutamate dehydrogenase in the presence of 2-oxoglutaric acid and NAD(P)H.
In a more preferable embodiment, the steps (a) and (b) are performed at the same time.
The present invention also is directed to a reagent kit for homocysteine determination comprising a thiol compound, a homocysteine-converting enzyme, a homocysteine cosubstrate, an SH reagent, and an oxidative color-developing agent.
In one preferable embodiment, the SH reagent is contained in another container from one for the thiol compound, the homocysteine-converting enzyme and the homocysteine cosubstrate.
In one preferable embodiment, the homocysteine-converting enzyme is contained in another container from one for the homocysteine cosubstrate.
In a more preferable embodiment, the homocysteine-converting enzyme is S-adenosyl-L-homocysteine hydrolase, and the homocysteine cosubstrate is adenosine.
In an even more preferable embodiment, the kit further includes adenosine deaminase, phosphoric acid, purine nucleoside phosphorylase, xanthine oxidase, and peroxidase.
In another preferable embodiment, the adenosine deaminase is contained in another container from one for the thiol compound, the S-adenosyl-L-homocysteine hydrolase and the adenosine.
In one preferable embodiment, the kit further includes uricase.
The present invention also is directed to a reagent kit for homocysteine determination comprising a thiol compound, a homocysteine-converting enzyme, a homocysteine cosubstrate, and a D-amino acid converting enzyme.
In one preferable embodiment, the kit further includes NAD(P)H, dehydrogenase using NAD(P)H as a coenzyme, and an ammonium salt or 2-oxo acid as its cosubstrate.
In a more preferable embodiment, the dehydrogenase is leucine dehydrogenase, and the cosubstrate of the enzyme is an ammonium salt.
In another more preferable embodiment, the dehydrogenase is glutamate dehydrogenase, and the cosubstrate of the enzyme is a 2-oxoglutaric acid.
In one preferable embodiment, the dehydrogenase is lactate dehydrogenase.
In another preferable embodiment, the homocysteine-converting enzyme is a methyltransferase using homocysteine as a methyl acceptor, and the homocysteine cosubstrate is a methyl donor.
In one preferable embodiment, the methyltransferase is homocysteine methyltransferase, and the methyl donor is D-methionine methyl sulfonium.
In one preferable embodiment, the D-amino acid converting enzyme is D-amino acid oxidase.
In one preferable embodiment, the D-amino acid oxidase is contained in another container from one for the thiol compound and the homocysteine methyltransferase.
In one preferable embodiment, the SH reagent is a maleimide derivative.